Label-free high-throughput optical technique for detecting biomolecular interactions

ABSTRACT

Methods and compositions are provided for detecting biomolecular interactions. The use of labels is not required and the methods can be performed in a high-throughput manner. The invention also provides optical devices useful as narrow band filters.

PRIORITY

This application claims the benefit of U.S. Provisional Application60/244,312, filed on Oct. 17, 2000, and U.S. Provisional Application60/283,314, filed Apr. 12, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of biosensors useful for detectingbiological material.

2. Background of the Art

Like DNA microarrays that are used to test and sequence DNA for theexpression of genes with massive parallelism, protein microarrays areexpected to become important tools for measuring protein interactions,determining the products of gene expression, and detecting for thepresence of protein analytes in solution. See, A. Lueking, et al.,“Protein Microarrays for Gene Expression and Antibody Screening,”Analytical Biochemistry 270, 103-111 (1999). Applications includemolecular biology research, pharmaceutical discovery, rational vaccinedevelopment, clinical sample screening for disease diagnosis,point-of-care diagnostic systems, and biological weapondetection/identification. See, M. Bourne, Business Opportunity Report,“Biosensors and Chemical Biosensors,” Business Communications Co. Inc.,(1999). For several applications, moderate readout system size and costcan be tolerated for a system that can provide thousands of parallelassays with extremely high microarray density using a disposable chipthat has very low cost. In addition, for some applications it isimportant to avoid the use of fluorescent molecule tags for labelingmicroarray-detected analytes due to the effect that the fluorophorattachment can have on the analyte's tertiary structure.

Several methods for building protein microarrays are being used byresearch groups and companies. Optical readout methods demonstrated forsingle (or <10-element) biosensors include “direct assays” (such assurface plasmon resonance, grating couplers, differencereflectometry/interferometry, resonant mirrors, and ellipsometry) and“indirect assays” (such as fluorescence spectroscopy, colormetricstaining, quantum dots, and upconverting phosphors). See, C. Striebel,et al., “Characterization of biomembranes by spectral ellipsometry,surface plasmon resonance, and interferometry with regard to biosensoroapplication,” Biosensors & Bioelectronics 9, 139-146 (1994). Of thesemethods, direct assays that do not require the addition of specialreagents to label detected analytes or other post-process chemicalamplification are preferred for assay simplicity. See, W. Huber, et al.,“Direct optical immunosensing (sensitivity and selectivity),” Sensorsand Actuators B6, 122-126 (1992). Direct assays are also useful fordetecting small protein molecules or structures that are not readilyattached to a label reagent. Of the direct methods, differencereflectometry and ellipsometry offer optical readout instrumentationthat is relatively simple to operate and interpret. See, A. Brecht, andG. Gauglitz, “Optical Probes and Transducers,” Biosensors &Bioelectronics 10, 923-936 (1995).

Biosensors have been developed to detect a variety of biomolecularcomplexes including oligonucleotides, antibody-antigen interactions,hormone-receptor interactions, and enzyme-substrate interactions. Ingeneral, biosensors consist of two components: a highly specificrecognition element and a transducer that converts the molecularrecognition event into a quantifiable signal. Signal transduction hasbeen accomplished by many methods, including fluorescence,interferometry, and gravimetry. See, A. Cunningham, “AnalyticalBiosensors.”

Of the optically-based transduction methods, direct methods that do notrequire labeling of analytes with fluorescent compounds are of interestdue to the relative assay simplicity and ability to study theinteraction of small molecules that are not readily labeled. Directoptical methods include surface plasmon resonance (SPR), gratingcouplers, ellipsometry, evanascent wave devices, and reflectometry.Theoretically predicted detection limits of these detection methods havebeen determined and experimentally confirmed to be feasible down todiagnostically relevant concentration ranges. However, to date, highlyparallel microarray approaches have not been applied to these methods.

The proposed invention utilizes a change in the refractive index upon asurface to determine when a chemically bound material is present withina specific location. Because both ellipsometry and reflectancespectrometry have been utilized as biosensors using similar phenomena,they will be described briefly so that the current disclosure can becompared.

Ellipsometry

Ellipsometry takes advantage of the different interaction of TM and TEmodes with thin films at reflection and radiation to measure opticalproperties (refractive index and thickness) of thin films. See, G. J.Pentti, et al., “A biosensor concept based on imaging ellipsometry forvisualization of biomolecular interactions,” Analytical Biochemistry232, 69-72 (1995). Parallel and perpendicular polarized light willexhibit different reflectivity and phase shift on reflection dependingon the optical constant of a semitransparent thin film. Changes inthickness below 5 angstroms can be resolved, giving this approach theability to resolve a protein monolayer. Ellipsometry is most commonlyused to measure film thickness of oxides and nitrides for semiconductorprocessing. While a single wavelength ellipsometer using a He:Ne lasercan be used to measure the refractive index and thickness of a singledeposited layer, spectroscopic ellipsometers can resolve several layerswithin an optical stack using a computer algorithm to fit measured dataacross a wavelength band. While the most basic ellipsometers probe asingle spot on a surface, imaging ellipsometers are now offered that canmeasure optical properties over a small area to create a map of opticalproperties.

Reflectance Interference Spectroscopy

Reflectance Interference Spectroscopy (RIS) is an extremely simplemethod for measuring protein adsorption onto optical surfaces. In RIS, asubstrate with a thin transparent film is illuminated with white light.Light reflected from the top and bottom surfaces of the thin filminterferes to generate a Fabry-Perot interference reflectance spectrumin which constructive interference occurs at some wavelengths whiledestructive interference occurs at others. The reflectance spectrumdepends upon the thickness and optical constant of the thin film. Likeellipsometry, RIS is typically used to measure oxide and nitride opticalthin film thickness on various substrates. The probe illumination andreflected signal may be applied with a fiber optic probe for large (˜2mm diameter) spot probing, or through a microscope objective for small(˜100 μm diameter) spot probing. Reflectance spectrum maps are usuallygenerated to monitor thin film thickness uniformity by operating thespectrometer with a scanning x-y stage. A single measurement can beperformed in <1 sec.

While theoretically able to measure layer thickness changes of severalangstroms, ellipsometry measurements of this sensitivity can bedifficult to interpret due to the degree of optical system alignmentthat is required to reproduce measurements. The readout system requiresa laser, a polarizer, and a rotating analyzer. Accurate instruments thatmeasure only a single location are expensive.

Instrumentation for reflectance spectrometry is inexpensive because themethod relies upon a white light source, a grating, and a linearphotodiode array, without any moving parts. Reflectance spectrometry istypically performed on optically flat surfaces. A computer model candetermine the adsorbed layer optical thickness that best matches thereflected spectrum. Layer thickness measurement resolution is limited bythe ability of the model to correctly fit small changes in the reflectedspectrum. Without the use of a resonant reflectance structure, thereflected spectrum of a flat surface covers a broad span of wavelengths.

BRIEF DESCRIPTION OF THE FIGURES

1. SEM Photo of an surface relief structure in photoresist showingterraced profile that is designed to reflect a narrow range ofwavelengths.

2. Graphic representation of how adsorbed material, such as a proteinmonolayer, will increase the reflected wavelength of a surface reliefstructure.

3. FIG. 3 illustrates a microarray biosensor.

4. Response as a function of wavelength of a granting structure withupon which BSA had been deposited at high concentration, measured inair. Before protein deposition, the resonant wavelength of the structureis 380 nm and is not observable with the instrument used for thisexperiment.

5. Response as a function of wavelength comparing an untreated gratingstructure with one upon which BSA had been deposited. Both measurementswere taken with water on the slide's surface.

6. Response as a function of wavelength of a grating structure with uponwhich borrelia bacteria had been deposited at high concentration,measured in water.

7. Optical structure used to simulate the effect of protein adsorptionon a resonant reflection grating.

8. Reflected intensity as a function of wavelength for a resonantgrating structure when various thickness of protein are incorporatedonto the upper surface.

9. Linear relationship between reflected wavelength and protein coatingthickness for the structure shown in FIG. 7.

10. A cross sectional diagram of a sensor that incorporates an ITOgrating.

11. An SEM photograph of a top view of the ITO grating of FIG. 10.

12. A diagram demonstrating that a single electrode can comprise aregion that contains many grating periods and that by building severalseparate grating regions on the same substrate surface, an array ofsensor electrodes can be created.

13. An SEM photograph showing the separate grating regions of FIG. 12.

14. A diagram showing a sensor chip upper surface immersed in a liquidsample so that an electrical potential can be applied to the sensor thatis capable of attracting or repelling a molecule near the electrodesurface.

15. A diagram depicting the attraction of electronegative molecules to asensor surface when a positive voltage is applied to an electrode.

16. A diagram demonstrating the application of a repelling force toelectronegative molecules using a negative electrode voltage.

17. A diagram of a microtiter plate incorporating a biosensor of theinvention.

18. A diagram of a biosensor or transmission filter comprising a set ofconcentric rings

19. A diagram of a biosensor or transmission filter comprising ahexagonal grid of holes or posts that approximates a concentric ringstructure.

FIG. 20A-B show schematic diagrams of one embodiment optical gratingstructure used for colormetric resonant reflectance biosensor.n_(substrate) represents substrate material. n₁ represents a coverlayer. n₂ represents a two-dimensional grating. n_(bio) represents oneor more specific binding substances. t₁ represents the thickness of thecover layer. t₂ represents the thickness of the grating. t_(bio)represents the thickness of the layer of one or more specific bindingsubstances. FIG. 20A shows a cross-sectional view of a biosensor. FIG.20B shows a diagram of a biosensor.

FIG. 21A-B shows a grating comprising a rectangular grid of squares(FIG. 21A) or holes (FIG. 21B).

FIG. 22 shows a schematic drawing of a linear grating structure.

FIG. 23 shows a biosensor cross-section profile in which an embossedsubstrate is coated with a higher refractive index material such as ZnSor SiN. A cover layer of, for example, epoxy or SOG is layered on top ofthe higher refractive index material and one or more specific bindingsubstances are immobilized on the cover layer.

FIG. 24 shows a biosensor cross-section profile utilizing a sinusoidallyvarying grating profile.

FIG. 25 shows three types of surface activation chemistry (Amine,Aldehyde, and Nickel) with corresponding chemical linker molecules thatcan be used to covalently attach various types of biomolecule receptorsto a biosensor.

FIG. 26A-C shows methods that can be used to amplify the mass of abinding partner such as detected DNA or detected protein on the surfaceof a biosensor.

SUMMARY OF THE INVENTION

This invention describes a biosensor which utilizes a surface that isengineered to reflect predominantly at a particular wavelength whenilluminated with broadband light. Biodetection is achieved when materialadsorbed onto the reflector's surface results in a shift in thereflected wavelength. The reflecting surface may contain either a singledetection region, or multiple detection regions that form a biosensormicroarray. While several one-dimensional engineered surfaces have beenshown to exhibit the ability to select a narrow range of reflected ortransmitted wavelengths from a broadband excitation source (thin filminterference filters and Bragg reflectors), the deposition of additionalmaterial onto their upper surface results only in a change in theresonance linewidth, rather than the resonance wavelength. By usingtwo-dimensional and three-dimensional grating structures, the ability toalter the reflected wavelength with the addition of material to thesurface is obtained. In this disclosure, the use of two such structuresfor reflected wavelength modulation by addition of biological materialto the upper surface are described. The first structure is athree-dimensional surface-relief volume diffractive grating, and thesecond structure is a two-dimensional structured surface. Biologicalmaterial may include DNA, protein, peptides, cells, bacteria, viruses.

In one embodiment, a biosensor having a reflective surface which has asculptured two or three dimensional surface coated with a reflectivematerial which reflects predominantly at a single wavelength whenilluminated with broadband light, and which reflects at a new wavelengthof light when biological matter is deposited or adsorbed upon thereflective surface. The reflective surface is a surface relief or volumediffractive structure having a two-dimensional grating. The reflectedwavelength is predominantly at a single wavelength due to the effect ofresonant scattering through a guided mode. Such biosensors areassociated with a light source which directs light to the reflectivesurface and a detector which detects light reflected from the reflectivesurface. The biosensor is typically used to detect biological mattersuch as DNA, proteins, peptides, cells, viruses, or bacteria.Preferably, the biosensor reflective surface is coated with an array ofdistinct locations containing specific binding substances to form amicroarray biosensor. The specific binding substance for example may beDNA, RNA, protein or polypeptide. The microarray has the distinctlocations defined as microarray spots of 50-500 microns in diameterpreferably 150-200 micron in diameter. The relief volume diffractionstructures are smaller than the resonant wavelength and are typicallyless than 1 micron in diameter.

A microarray sensor is made of a sheet material having a first andsecond surface wherein the first surface defines relief volumediffraction structures coated with a reflective material such as silveror gold and an array of distinct locations on the first surface iscoated with a specific binding substance such as DNA, RNA,oligonucleotide, protein or polypeptide. In this embodiment there is ashift in wave length of the reflected light when a specific bindingsubstance is bound to its binding partner. In another embodiment thebiosensor is made of transparent material having a two or threedimensional sculpture light receiving surface and a light emittingsurface which emits light at the light emitting surface primarily at asingle wavelength when illuminated on the light receiving surface withbroadband light and which emits light at a different wave length whenbiological matter is deposited on the light receiving surface. In thisembodiment the biosensor associated with a light source which directslight to the light receiving surface and a detector which receives lightfrom the light emitting surface. The light transmitting biosensorembodiment has a sculptured two or three dimensional surface designed totransmit predominantly at a single wavelength when illuminated withbroadband light, and which transmits at a new wavelength of light whenbiological matter is deposited on the sculpture. This light transmittingembodiment may be formed into a microsensor array as described above.

DETAILED DESCRIPTION OF THE INVENTION

The proposed method is similar to reflectance spectrometry, except thatrather than a flat optical surface, a grating surface is used to definea very narrow range of reflected wavelength. Because the reflectedwavelength is confined to a narrow bandwidth, very small changes in theoptical characteristics of the surface manifest themselves in easilyobserved changes in reflected wavelength spectra. The narrow reflectionbandwidth provides a surface adsorption sensitivity advantage comparedto reflectance spectrometry on a flat surface. The method can use thesame readout instrumentation as reflectance spectrometry on a flatsurface, which is less expensive than ellipsometer based detection, andtherefore has a system cost/complexity advantage over ellipsometry-baseddetection. Because detection by the proposed system can be engineered toresult in an easily observable change in reflected color (i.e. bluereflection corresponds to no absorption, while green reflectioncorresponds to a positive signal), it may be possible to furthersimplify the readout instrumentation by the application of a filter sothat only positive results over a determined threshold trigger adetection.

A second advantage of the proposed approach is the cost of producing thegrating surface. Once a metal master plate has been produced, thegrating can be mass-produced very inexpensively by stamping thestructure into a plastic material like vinyl. After stamping, thegrating surface is made reflective by blanket deposition of a thin metalfilm such as gold, silver, or aluminum. Compared to MEMS-basedbiosensors that rely upon photolithography, etching, and wafer bondingprocedures, the proposed structure is very inexpensive. It is estimatedthat the cost of 1 cm² gratings will be ˜$0.02.

Unlike surface plasmon resonance, resonant mirrors, and waveguidebiosensors, the proposed method enables many thousands of individualbinding reactions to take place simultaneously upon the sensor surface.Readout of the reflected color can be performed serially by focusing amicroscope objective onto individual microarray spots and reading thereflected spectrum, or in parallel by projecting the reflected image ofthe microarray onto a high resolution color CCD camera.

Surface-Relief Volume Diffractive Biosensor

In one embodiment, a surface-relief volume diffractive structure is usedas a microarray biosensor for detecting the adsorption of smallquantities of materials onto surfaces. The theory describing the designand fabrication of such structures has been published. See, J. J. Cowen,“Aztec surface-relief volume diffractive structure,” J. Opt. Soc. Am. A,Vol. 7, No. 8, August, 1990. While the use of these structures foroptical display and telecommunication filters is being pursued, thisinvention pertains to their first use as a biosensor.

The surface relief volume diffractive grating structure is athree-dimensional phase-quantized terraced surface relief pattern whosegroove pattern resembles a stepped pyramid, as shown in FIG. 1. When thegrating is illuminated by a beam of broadband radiation, light will becoherently reflected from the equally spaced terraces at a wavelengthgiven by twice the step spacing times the index of refraction of thesurrounding medium. The structure can be replicated by metal masteringand molding into plastic in the same manner as conventional embossedsurface relief elements. Light of a given wavelength is resonantlydiffracted or reflected from the steps that are a half-wavelength apart,and with a bandwidth that is inversely proportional to the number ofsteps. The reflected or diffracted color can be controlled by thedeposition of a dielectric layer so that a new wavelength is selected,depending on the index of refraction of the coating.

The desired stepped-phase structure is first produced in photoresist bycoherently exposing a thin photoresist film to three laser beams, asdescribed in previous research. See, J. J. Cowen, “The recording andlarge scale replication of crossed holographic grating arrays usingmultiple beam interferometry,” in International Conference on theApplication, Theory, and Fabrication of Periodic Structures, DiffractionGratings, and Moire Phenomena II, J. M. Lerner, ed., Proc. Soc.Photo-Opt. Instrum. Eng., 503, 120-129, 1984; J. J. Cowen, “Holographichoneycomb microlens,” Opt. Eng. 24, 796-802 (1985); J. J. Cowen and W.D. Slafer, “The recording and replication of holographic micropatternsfor the ordering of photographic emulsion grains in film systems,” J.Imaging Sci. 31, 100-107, 1987. The nonlinear etching characteristics ofphotoresist are used to develop the exposed film to create a threedimensional relief pattern. The photoresist structure is then replicatedusing standard embossing procedures. First, a thin silver film isdeposited over the photoresist structure to form a conducting layer uponwhich a thick film of nickel may be electroplated. The nickel “master”plate is then used to emboss directly into a plastic film, such asvinyl, that has been softened by heating or solvent.

The color that is reflected from the terraced step structure istheoretically given as twice the step height times the index ofrefraction of the overcoating dielectric layer. To use this type ofstructure as a biosensor, a thin film of adsorbed molecules provides adifferent refractive index on the surface of the grating than thesurrounding medium. Thus, when the molecules incorporate themselves intothe diffractive structure, they cause a change in the color that isreflected or diffracted from the grating. Proteins films reportedly haveindices of refraction of 1.4 to 1.5, and therefore can providesubstantial shift in the reflected or diffracted spectrum, even for afilm that is less than 1000 nm thickness. It is expected that thickmolecular films can be differentiated from thin molecular films by theextent of wavelength shift as well as the strength of the reflected ordiffracted order.

Two-Dimensional Structured Surface Biosensor

In one embodiment of the invention, a subwavelength structured surface(SWS) is used to create a sharp optical resonant reflection at aparticular wavelength that can be tracked with high sensitivity asbiological materials, such as specific binding substances or bindingpartners or both, are attached to a colormetric resonant diffractivegrating surface that acts as a surface binding platform.

Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J. Opt.Soc. Am. A, Vol. 13, No. 5, p. 993, May; Magnusson, & Want, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August, 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). An SWS structure contains asurface-relief, two-dimensional grating in which the grating period issmall compared to the wavelength of incident light so that nodiffractive orders other than the reflected and transmitted zerothorders are allowed to propagate. A SWS surface narrowband filter cancomprise a two-dimensional grating sandwiched between a substrate layerand a cover layer that fills the grating grooves. Optionally, a coverlayer is not used. When the effective index of refraction of the gratingregion is greater than the substrate or the cover layer, a waveguide iscreated. When a filter is designed properly, incident light passes intothe waveguide region and propagates as a leaky mode. A grating structureselectively couples light at a narrow band of wavelengths into thewaveguide. The light propagates only a very short distance (on the orderof 10-100 micrometers), undergoes scattering, and couples with theforward- and backward-propagating zeroth-order light. This highlysensitive coupling condition can produce a resonant grating effect onthe reflected radiation spectrum, resulting in a narrow band ofreflected or transmitted wavelengths. The depth and period of thetwo-dimensional grating are less than the wavelength of the resonantgrating effect.

The reflected or transmitted color of this structure can be modulated bythe addition of molecules such as specific binding substances or bindingpartners or both to the upper surface of the cover layer or the gratingsurface. The added molecules increase the optical path length ofincident radiation through the structure, and thus modify the wavelengthat which maximum reflectance or transmittance will occur.

A schematic diagram of an example of a SWS structure is shown in FIG.20. In FIG. 20, n_(substrate) represents a substrate material. N₁represents an optional cover layer. N₂ represents a two-dimensionalgrating. N_(bio) represents one or more specific binding substances. t₁represents the thickness of the cover layer. t₂ represents the thicknessof the grating. t_(bio) represents the thickness of the layer of one ormore specific binding substances. In one embodiment, are n2>n1 (see FIG.20). Layer thicknesses (i.e. cover layer, one or more specific bindingsubstances, or a two-dimensional grating)) are selected to achieveresonant wavelength sensitivity to additional molecules on the topsurface The grating period is selected to achieve resonance at a desiredwavelength.

In one embodiment, a biosensor, when illuminated with white light, isdesigned to reflect only a single wavelength. When specific bindingsubstances are attached to the surface of the biosensor, the reflectedwavelength (color) is shifted due to the change of the optical path oflight that is coupled into the grating. By linking specific bindingsubstances to a biosensor surface, complementary binding partnermolecules can be detected without the use of any kind of fluorescentprobe or particle label. The detection technique is capable of resolvingchanges of, for example, ˜0.1 nm thickness of protein binding, and canbe performed with the biosensor surface either immersed in fluid ordried.

A detection system consists of, for example, a light source thatilluminates a small spot of a biosensor at normal incidence through, forexample, a fiber optic probe, and a spectrometer that collects thereflected light through, for example, a second fiber optic probe also atnormal incidence. Because no physical contact occurs between theexcitation/detection system and the biosensor surface, no specialcoupling prisms are required and the biosensor can be easily adapted toany commonly used assay platform including, for example, microtiterplates and microarray slides. A single spectrometer reading can beperformed in several milliseconds, thus it is possible to quicklymeasure a large number of molecular interactions taking place inparallel upon a biosensor surface, and to monitor reaction kinetics inreal time.

This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

One embodiment of the invention provides a SWS biosensor. A SWSbiosensor comprises a two-dimensional grating, a substrate layer thatsupports the two-dimensional grating, and one or more specific bindingsubstances immobilized on the surface of the two-dimensional gratingopposite of the substrate layer.

A two-dimensional grating can be comprised of a material, including, forexample, zinc sulfide, titanium dioxide, and silicon nitride. Across-sectional profile of a two-dimensional grating can comprise anyperiodically repeating function, for example, a “square-wave.” Atwo-dimensional grating can be comprised of a repeating pattern ofshapes selected from the group consisting of squares, circles, ellipses,triangles, trapezoids, sinusoidal waves, ovals, rectangles, andhexagons. A sinusoidal cross-sectional profile is preferable formanufacturing applications that require embossing of a grating shapeinto a soft material such as plastic.

Linear (i.e., rectangular) gratings have resonant characteristics wherethe illuminating light polarization is oriented perpendicular to thegrating period. However, a hexagonal grid of holes has betterpolarization symmetry than a rectangular grid of holes. Therefore, acolorimetric resonant reflection biosensor of the invention cancomprise, for example, a hexagonal array of holes (see FIG. 21B) or agrid of parallel lines (see FIG. 21A). A linear grating has the samepitch (i.e. distance between regions of high and low refractive index),period, layer thicknesses, and material properties as the hexagonalarray grating. However, light must be polarized perpendicular to thegrating lines in order to be resonantly coupled into the opticalstructure. Therefore, a polarizing filter oriented with its polarizationaxis perpendicular to the linear grating must be inserted between theillumination source and the biosensor surface. Because only a smallportion of the illuminating light source is correctly polarized, alonger integration time is required to collect an equivalent amount ofresonantly reflected light compared to a hexagonal grating.

While a linear grating can require either a higher intensityillumination source or a longer measurement integration time compared toa hexagonal grating, the fabrication requirements for the linearstructure are simpler. A hexagonal grating pattern is produced byholographic exposure of photoresist to three mutually interfering laserbeams. The three beams are precisely aligned in order to produce agrating pattern that is symmetrical in three directions. A lineargrating pattern requires alignment of only two laser beams to produce aholographic exposure in photoresist, and thus has a reduced alignmentrequirement. A linear grating pattern can also be produced by, forexample, direct writing of photoresist with an electron beam. Also,several commercially available sources exist for producing lineargrating “master” templates for embossing a grating structure intoplastic. A schematic diagram of a linear grating structure is shown inFIG. 22

A rectangular grid pattern can be produced in photoresist using anelectron beam direct-write exposure system. A single wafer can beilluminated as a linear grating with two sequential exposures with thepart rotated 90-degrees between exposures.

A two-dimensional grating can also comprise, for example, a “stepped”profile, in which high refractive index regions of a single, fixedheight are embedded within a lower refractive index cover layer. Thealternating regions of high and low refractive index provide an opticalwaveguide parallel to the top surface of the biosensor. See FIG. 23.

For manufacture, a stepped structure is etched or embossed into asubstrate material such as glass or plastic. See FIG. 23. A uniform thinfilm of higher refractive index material, such as silicon nitride orzinc sulfide is deposited on this structure. The deposited layer willfollow the shape contour of the embossed or etched structure in thesubstrate, so that the deposited material has a surface relief profilethat is identical to the original embossed or etched profile. Thestructure can be completed by the application of an optional cover layercomprised of a material having a lower refractive index than the higherrefractive index material and having a substantially flat upper surface.The covering material can be, for example, glass, epoxy, or plastic.

This structure allows for low cost biosensor manufacturing, because itcan be mass produced. A “master” grating can be produced in glass,plastic, or metal using, for example, a three-beam laser holographicpatterning process. A master grating can be repeatedly used to emboss aplastic substrate. The embossed substrate is subsequently coated with ahigh refractive index material and optionally, a cover layer.

While a stepped structure is simple to manufacture, it is also possibleto make a resonant biosensor in which the high refractive index materialis not stepped, but which varies with lateral position. FIG. 24 shows aprofile in which the high refractive index material of thetwo-dimensional grating, n₂, is sinusoidally varying in height. Toproduce a resonant reflection at a particular wavelength, the period ofthe sinusoid is identical to the period of an equivalent steppedstructure. The resonant operation of the sinusoidally varying structureand its functionality as a biosensor has been verified using GSOLVER(Grating Solver Development Company, Allen, Tex., USA) computer models.

Techniques for making two-dimensional gratings are disclosed in J. Opt.Soc. Am No. 8, August 1990, pp. 1529-44. Biosensors of the invention canbe made in, for example, a semiconductor microfabrication facility.Biosensors can also be made on a plastic substrate using continuousembossing and optical coating processes. For this type of manufacturingprocess, a “master” structure is built in a rigid material such as glassor silicon, and is used to generate “mother” structures in an epoxy orplastic using one of several types of replication procedures. The“mother” structure, in turn, is coated with a thin film of conducivematerial, and used as a mold to electroplate a thick film of nickel. Thenickel “daughter” is released from the plastic “mother” structure.Finally, the nickel “daughter” is bonded to a cylindrical drum, which isused to continuously emboss the surface relief structure into a plasticfilm. A device structure that uses an embossed plastic substrate isshown in FIG. 23. Following embossing, the plastic structure isovercoated with a thin film of high refractive index material, andoptionally coated with a planarizing, cover layer polymer, and cut toappropriate size.

A substrate for a SWS biosensor can comprise, for example, glass,plastic or epoxy. Optionally, a substrate and a two-dimensional gratingcan comprise a single unit. That is, a two dimensional grating andsubstrate are formed from the same material, for example, glass,plastic, or epoxy. The surface of a single unit comprising thetwo-dimensional grating is coated with a material having a highrefractive index, for example, zinc sulfide, titanium dioxide, andsilicon nitride. One or more specific binding substances can beimmobilized on the surface of the material having a high refractiveindex or on an optional cover layer.

A biosensor of the invention can further comprise a cover layer on thesurface of a two-dimensional grating opposite of a substrate layer.Where a cover layer is present, the one or more specific bindingsubstances are immobilized on the surface of the cover layer opposite ofthe two-dimensional grating. Preferably, a cover layer comprises amaterial that has a lower refractive index than a material thatcomprises the two-dimensional grating. A cover layer can be comprisedof, for example, glass (including spin-on glass (SOG)), epoxy, orplastic.

For example, various polymers that meet the refractive index requirementof a biosensor can be used for a cover layer. SOG can be used due to itsfavorable refractive index, ease of handling, and readiness of beingactivated with specific binding substances using the wealth of glasssurface activation techniques. When the flatness of the biosensorsurface is not an issue for a particular system setup, a gratingstructure of SiN/glass can directly be used as the sensing surface, theactivation of which can be done using the same means as on a glasssurface.

Resonant reflection can also be obtained without a planarizing coverlayer over a two-dimensional grating. For example, a biosensor cancontain only a substrate coated with a structured thin film layer ofhigh refractive index material. Without the use of a planarizing coverlayer, the surrounding medium (such as air or water) fills the grating.Therefore, specific binding substances are immobilized to the biosensoron the tops, bottoms, and sides of a two-dimensional grating, ratherthan only on an upper surface.

In general, a biosensor of the invention will be illuminated with whitelight that will contain light of every polarization angle. Theorientation of the polarization angle with respect to repeating featuresin a biosensor grating will determine the resonance wavelength. Forexample, a “linear grating” biosensor structure consisting of a set ofrepeating lines and spaces will have two optical polarizations that cangenerate separate resonant reflections. Light that is polarizedperpendicularly to the lines is called “s-polarized,” while light thatis polarized parallel to the lines is called “p-polarized.” Both the sand p components of incident light exist simultaneously in an unfilteredillumination beam, and each generates a separate resonant signal. Abiosensor structure can generally be designed to optimize the propertiesof only one polarization (the s-polarization), and the non-optimizedpolarization is easily removed by a polarizing filter.

In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used that consists of a set ofconcentric rings. In this structure, the difference between the insidediameter and the outside diameter of each ring is equal to one-half of agrating period. Each successive ring has an inside diameter that is onegrating period greater than the inside diameter of the previous ring.The concentric ring pattern extends to cover a single sensorlocation—such as a microarray spot or a microtiter plate well. Eachseparate microarray spot or microtiter plate well has a separateconcentric ring pattern centered within it. See FIG. 18. Allpolarization directions of such a structure have the samecross-sectional profile. The concentric ring structure must beilluminated precisely on-center to preserve polarization independence.The grating period of a concentric ring structure is less than thewavelength of the resonantly reflected light. In general, the gratingperiod is less than one micron. The grating height is also less than thewavelength of light, and is generally less than one micron.

In another embodiment, a hexagonal grid of holes (or a hexagonal grid ofposts) closely approximates the concentric circle structure withoutrequiring the illumination beam to be centered upon any particularlocation of the grid. See FIG. 19. Such a hexagonal grid pattern isautomatically generated by the optical interference of three laser beamsincident on a surface from three directions at equal angles. In thispattern, the holes (or posts) are centered upon the corners of an arrayof closely packed hexagons as shown in FIG. 19. Such a hexagonal gridhas three polarization directions that “see” the same cross-sectionalprofile. The hexagonal grid structure, therefore, provides equivalentresonant reflection spectra using light of any polarization angle. Thus,no polarizing filter is required to remove unwanted reflected signalcomponents.

The invention provides a resonant reflection structure and transmissionfilter structures comprising concentric circle gratings and hexagonalgrids of holes or posts. For a resonant reflection structure, lightoutput is measured on the same side of the structure as the illuminatinglight beam. For a transmission filter structure, light output ismeasured on the opposite side of the structure as the illuminating beam.The reflected and transmitted signals are complementary. That is, if awavelength is strongly reflected, it is weakly transmitted. Assuming noenergy is absorbed in the structure itself, the reflected+transmittedenergy at any given wavelength is constant. The resonant reflectionstructure and transmission filters are designed to give a highlyefficient reflection at a specified wavelength. Thus, a reflectionfilter will “pass” a narrow band of wavelengths, while a transmissionfilter will “cut” a narrow band of wavelengths from incident light.

A resonant reflection structure or a transmission filter structure cancomprising a two-dimensional grating arranged in a pattern of concentriccircles. A resonant reflection structure or transmission filterstructure can also comprise a hexagonal grid of holes or posts. Whenthese structure are illuminated with an illuminating light beam, areflected radiation spectrum is produced that is independent of anillumination polarization angle of the illuminating light beam. Whenthese structures are illuminated a resonant grating effect is producedon the reflected radiation spectrum, wherein the depth and period of thetwo-dimensional grating or hexagonal grid of holes or posts are lessthan the wavelength of the resonant grating effect. These structuresreflect a narrow band of light is reflected from the structure when thestructure is illuminated with a broadband of light. Resonant reflectionstructures and transmission filter structures of the invention can beused as biosensors. For example, one or more specific binding substancescan be immobilized on the hexagonal grid of holes or posts or on thetwo-dimensional grating arranged in concentric circles.

Specific Binding Substances and Binding Partners

One or more specific binding substances are immobilized on thetwo-dimensional grating or cover layer, if present, by for example,physical adsorption or by chemical binding. A specific binding substancecan be, for example, a nucleic acid, polypeptide, antigen, polyclonalantibody, monoclonal antibody, single chain antibody (scFv), F(ab)fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule, cell,virus, bacteria, or biological sample. A biological sample can be forexample, blood, plasma, serum, gastrointestinal secretions, homogenatesof tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, or prostatitc fluid.

Preferably, one or more specific binding substances are arranged in amicroarray of distinct locations on a biosensor. A microarray ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor of the invention such that asurface contains many distinct locations, each with a different specificbinding substance or with a different amount of a specific bindingsubstance. For example, an array can comprise 1, 10, 100, 1,000, 10,000,or 100,000 distinct locations. Such a biosensor surface is called amicroarray because one or more specific binding substances are typicallylaid out in a regular grid pattern in x-y coordinates. However, amicroarray of the invention can comprise one or more specific bindingsubstance laid out in any type of regular or irregular pattern. Forexample, distinct locations can define a microarray of spots of one ormore specific binding substances. A microarray spot can be about 50 toabout 500 microns in diameter. A microarray spot can also be about 150to about 200 microns in diameter. One or more specific bindingsubstances can be bound to their specific binding partners.

A microarray on a biosensor of the invention can be created by placingmicrodroplets of one or more specific binding substances onto, forexample, an x-y grid of locations on a two-dimensional grating or coverlayer surface. When the biosensor is exposed to a test sample comprisingone or more binding partners, the binding partners will bepreferentially attracted to distinct locations on the microarray thatcomprise specific binding substances that have high affinity for thebinding partners. Some of the distinct locations will gather bindingpartners onto their surface, while other locations will not.

A specific binding substance specifically binds to a binding partnerthat is added to the surface of a biosensor of the invention. A specificbinding substance specifically binds to its binding partner, but doesnot substantially bind other binding partners added to the surface of abiosensor. For example, where the specific binding substance is anantibody and its binding partner is a particular antigen, the antibodyspecifically binds to the particular antigen, but does not substantiallybind other antigens. A binding partner can be, for example, a nucleicacid, polypeptide, antigen, polyclonal antibody, monoclonal antibody,single chain antibody (scFv), F(ab) fragment, F(ab′)₂ fragment, Fvfragment, small organic molecule, cell, virus, bacteria, and biologicalsample. A biological sample can be, for example, blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, and prostatitc fluid.

One example of a microarray of the invention is a nucleic acidmicroarray, in which each distinct location within the array contains adifferent nucleic acid molecule. In this embodiment, the spots withinthe nucleic acid microarray detect complementary chemical binding withan opposing strand of a nucleic acid in a test sample.

While microtiter plates are the most common format used for biochemicalassays, microarrays are increasingly seen as a means for maximizing thenumber of biochemical interactions that can be measured at one timewhile minimizing the volume of precious reagents. By application ofspecific binding substances with a microarray spotter onto a biosensorof the invention, specific binding substance densities of 10,000specific binding substances/in² can be obtained. By focusing anillumination beam to interrogate a single microarray location, abiosensor can be used as a label-free microarray readout system.

Immobilization or One or More Specific Binding Substances

Immobilization of one or more binding substances onto a biosensor isperformed so that a specific binding substance will not be washed awayby rinsing procedures, and so that its binding to binding partners in atest sample is unimpeded by the biosensor surface. Several differenttypes of surface chemistry strategies have been implemented for covalentattachment of specific binding substances to, for example, glass for usein various types of microarrays and biosensors. These same methods canbe readily adapted to a biosensor of the invention. Surface preparationof a biosensor so that it contains the correct functional groups forbinding one or more specific binding substances is an integral part ofthe biosensor manufacturing process.

One or more specific binding substances can be attached to a biosensorsurface by physical adsorption (i.e., without the use of chemicallinkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on a biosensor surface and provide definedorientation and conformation of the surface-bound molecules.

Types of chemical binding include, for example, amine activation,aldehyde activation, and nickel activation. These surfaces can be usedto attach several different types of chemical linkers to a biosensorsurface, as shown in FIG. 25. While an amine surface can be used toattach several types of linker molecules, an aldehyde surface can beused to bind proteins directly, without an additional linker. A nickelsurface can be used to bind molecules that have an incorporatedhistidine (“his”) tag. Detection of “his-tagged” molecules with anickel-activated surface is well known in the art (Whitesides, Anal.Chem. 68, 490).

For the detection of binding partners at concentrations less than about˜0.1 ng/ml, it is preferable to amplify and transduce binding partnersbound to a biosensor into an additional layer on the biosensor surface.The increased mass deposited on the biosensor can be easily detected asa consequence of increased optical path length. By incorporating greatermass onto a biosensor surface, the optical density of binding partnerson the surface is also increased, thus rendering a greater resonantwavelength shift than would occur without the added mass. The additionof mass can be accomplished, for example, enzymatically, through a“sandwich” assay, or by direct application of mass to the biosensorsurface in the form of appropriately conjugated beads or polymers ofvarious size and composition. This principle has been exploited forother types of optical biosensors to demonstrate sensitivity increasesover 1500× beyond sensitivity limits achieved without massamplification. See, e.g., Jenison, et al., “Interference-based detectionof nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19: 62-65, 2001.

As an example, FIG. 26A shows that an NH₂-activated biosensor surfacecan have a specific binding substance comprising a single-strand DNAcapture probe immobilized on the surface. The capture probe interactsselectively with its complementary target binding partner. The bindingpartner, in turn, can be designed to include a sequence or tag that willbind a “detector” molecule. As shown in FIG. 26A, a detector moleculecan contain, for example, a linker to horseradish peroxidase (HRP) that,when exposed to the correct enzyme, will selectively deposit additionalmaterial on the biosensor only where the detector molecule is present.Such a procedure can add, for example, 300 angstroms of detectablebiomaterial to the biosensor within a few minutes.

A “sandwich” approach can also be used to enhance detection sensitivity.In this approach, a large molecular weight molecule can be used toamplify the presence of a low molecular weight molecule. For example, abinding partner with a molecular weight of, for example, 1 kDa, can betagged with, for example, SMPT, DMP, NNDC, histidine, or a biotinmolecule, as shown in FIG. 26B. Where the tag is biotin, the biotinmolecule will binds strongly with streptavidin, which has a molecularweight of 60 kDa. Because the biotin/streptavidin interaction is highlyspecific, the streptavidin amplifies the signal that would be producedonly by the small binding partner by a factor of 60.

Detection sensitivity can be further enhanced through the use ofchemically derivatized small particles. “Nanoparticles” made ofcolloidal gold, various plastics, or glass with diameters of about 3-300nm can be coated with molecular species that will enable them tocovalently bind selectively to a binding partner. For example, as shownin FIG. 26C, nanoparticles that are covalently coated with streptavidincan be used to enhance the visibility of biotin-tagged binding partnerson the biosensor surface. While a streptavidin molecule itself has amolecular weight of 60 kDa, the derivatized bead can have a molecularweight of, for example, 60 KDa. Binding of a large bead will result in alarge change in the optical density upon the biosensor surface, and aneasily measurable signal. This method can result in an approximately1000× enhancement in sensitivity resolution.

Description of a Microarray

One method for implementing parallel chemical affinity analysis is toplace chemical reagents onto a planar solid support, such that the solidsupport contains many individual locations, each with a differentchemical reagent. Such an activated planar solid support is called a“microarray” because the different chemical reagents are typically laidout in a regular grid pattern in x-y coordinates. One possibleimplementation is a DNA microarray, in which each individual locationwithin the array contains a different sequence of oligonucleotides. Inthis embodiment, the spots within the DNA microarray detectcomplementary chemical binding with an opposing strand of DNA in a testsample. In order to detect the presence of the opposing DNA when itbinds, the opposing DNA is “tagged” with a fluorophor, so that itspresence will be indicated by the light emitted by the fluorophor whenthe microarray location is excited with a laser. The original DNA thatis placed onto the microarray is an Affinity Ligand Reagent (ALR) thathas high affinity for its complementary DNA sequence, and low affinityfor all other DNA sequences.

Protein Microarray

While the DNA microarray is used to detect and sequence the DNAcomponents of a test sample, a protein microarray would be used todetect the affinity interaction between proteins that are placed ontothe individual microarray locations, and proteins within a testsolution. For example, by placing individual protein antibodies ontodifferent locations on a microarray surface, it is possible to detectthe corresponding antigens in a test sample when they bind selectivelyto the protein antibodies. Like the DNA microarrays, the detectedprotein may be labeled with a fluorophor to enable detection.Alternatively, some other form of molecular or particle tag may be boundto the detected protein to signal its presence on the microarraysurface.

Many methods have been developed to detect the presence of bound proteinanalyte without any type of label, including methods that result indetection of modification of mass, refractive index, or surfaceroughness on the microarray surface. Several methods will be brieflyreviewed in the “Competing Technology” section of this disclosure.

Resonant Reflection Microarray

To build a microarray biosensor, a grating designed to reflectpredominantly at a particular wavelength is required. Techniques formaking such gratings are disclosed in J. Opt. Soc. Am No. 8, August1990, pp. 1529-44. This reference is incorporated herein by reference. Amicroarray sensor would be created by placing microdroplets of highaffinity chemical receptor reagents onto an x-y grid of locations on thegrating surface. When the chemically active microarray is exposed to ananalyte, molecules will be preferentially attracted to microarraylocations that have high affinity. Some microarray locations will gatheradditional material onto their surface, while other locations will not.By measuring the shift in resonant wavelength within each individualmicroarray grating location, it is possible to determine which locationshave attracted additional material. The extent of the shift can be usedto determine the amount of bound analyte in the sample and the chemicalaffinity between the microarray receptor reagents and the analyte.

As shown in FIG. 1, each inverted pyramid is approximately 1 micron indiameter, and pyramid structures can be close-packed, a typicalmicroarray spot with a diameter of 150-200 microns can incorporateseveral hundred structures. FIG. 2 describes how individual microarraylocations (with an entire microarray spot incorporating hundreds ofpyramids now represented by a single pyramid for one microarray spot)can be optically queried to determine if material is adsorbed onto thesurface. When the structure is illuminated with white light, structureswithout significant bound material will reflect wavelengths determinedby the step height of the structure. When higher refractive indexmaterial, such as protein monolayers, are incorporated over thereflective metal surface, the reflected wavelength is modified to shifttoward longer wavelengths.

FIG. 3 is a schematic representation of a 9-element microarraybiosensor. Many individual grating structures, represented by smallgreen circles, lie within each microarray spot. The microarray spots,represented by the larger circles, will reflect white light in air at awavelength that is determined by the refractive index of material ontheir surface. Microarray locations with additional adsorbed materialwill have reflected wavelengths that are shifted toward longerwavelengths, represented by the larger circles.

Methods for Analysis of a Large Number of Protein Interactions inParallel

The biosensors of the invention can be used to study a large number ofprotein interactions in parallel. For example, in proteomics research,investigators determine the extent to which different groups of proteinsinteract, and the biosensors of the invention can be used to study theinteractions.

In the “protein chip” approach of using the biosensors of the invention,a variety of “bait” proteins, for example, antibodies, can beimmobilized in an array format onto the biosensors of the invention. Thesurface is then probed with the sample of interest and only the proteinsthat bind to the relevant bait proteins remain bound to the chip. Suchan approach is essentially a large-scale version of enzyme-linkedimmunosorbent assays. As described previously, the biosensor of thisinvention is designed to detect the adsorption of protein in a sample toa protein “bait” on the chip surface without the use of an enzyme orfluorescent label.

Several different types of assays can be performed to detect or measureprotein-protein interactions. For example, the protein chip surface cancomprise immobilized recombinant proteins, protein domains, orpolypeptides. A sample, for example, of cell lysates containing putativeinteraction partners are applied to the protein chip, followed bywashing to remove unbound material. Ideally, the bound proteins areeluted from the chip and identified by mass spectrometry. Optionally, aphage DNA display library can be applied to the chip followed by washingand amplification steps to isolate individual phage particles. Theinserts in these phage particles can then be sequenced to determine theidentity of the interacting partners.

For the above applications, and in particular proteomics applications,the ability to selectively bind material from a sample onto a proteinchip (or microarray) surface, followed by the ability to selectivelyremove bound material from one protein chip spot at a time for furtheranalysis is preferable. The biosensors of the invention are capable ofdetecting and quantifying the amount of protein from a sample that isbound to a biosensor array location. A modification to the basic sensorstructure can further enable the biosensor array to selectively attractor repel bound biological material from individual array locations.

As is well known in the art, an electromotive force can be applied tobiological molecules such as DNA, proteins, and peptides by subjectingthem to an electric field. The basic techniques of gel electrophoresisand capillary electrophoresis operate by application of an electricfield across a medium that contains DNA or protein molecules. Becausethese molecules are electronegative, they are attracted to a positivelycharged electrode and repelled by a negatively charged electrode.

A grating structure of the resonant optical biosensor can be built usingan electrically conducting material rather than an electricallyinsulating material. An electric field can be applied near the biosensorsurface. Where a grating operates as both a resonant reflector biosensorand as an electrode, the grating must comprise a material that is bothoptically transparent near the resonant wavelength, and has lowresistivity. In a preferred embodiment of the invention, the material isindium tin oxide, InSn_(x)O_(1-x) (ITO). ITO is commonly used to producetransparent electrodes for flat panel optical displays, and is thereforereadily available at low cost on large glass sheets. The refractiveindex of ITO can be adjusted by controlling x, the fraction of Sn thatis present in the material. Because the liquid test solution will havemobile ions (and will therefore be an electrical conductor) it isnecessary for the ITO electrodes to be coated with an insulatingmaterial. For the resonant optical biosensor, a grating layer must becoated with a layer with lower refractive index. Materials such as curedphotoresist (n=1.65), cured optical epoxy (n=1.5), and glass (n=1.4-1.5)are strong electrical insulators that also have a refractive index thatis lower than ITO (n=2.0-2.65). A cross sectional diagram of a sensorthat incorporates an ITO grating is shown in FIG. 10. An SEM photographof a top view of the ITO grating is shown in FIG. 11. As shown in FIG.11, a grating can be a continuous sheet of ITO that contains an array ofregularly spaced holes. The holes are filled in with an electricallyinsulating material, such as cured photoresist. The electricallyinsulating layer overcoats the ITO grating so that the upper surface ofthe structure is completely covered with electrical insulator, and sothat the upper surface is flat.

As shown in FIG. 12 and FIG. 13, a single electrode can comprise aregion that contains many grating periods. Building several separategrating regions on the same substrate surface creates an array of sensorelectrodes. Electrical contact to each sensor electrode is providedusing an electrically conducting trace that is built from the samematerial as the conductor within the sensor electrode (ITO). Theconducting trace is connected to a voltage source that can apply anelectrical potential to the electrode. To apply an electrical potentialto the sensor that is capable of attracting or repelling a molecule nearthe electrode surface, the sensor chip upper surface can be immersed ina liquid sample as shown in FIG. 14. A “common” electrode can be placedwithin the sample liquid, and a voltage can be applied between oneselected sensor electrode region and the common electrode. In this way,one, several, or all electrodes can be activated or inactivated at agiven time. FIG. 15 illustrates the attraction of electronegativemolecules to the sensor surface when a positive voltage is applied tothe electrode, while FIG. 16 illustrates the application of a repellingforce to electronegative molecules using a negative electrode voltage.

Microtiter Plate Implementation

While a microarray format enables many individual “bait” probes (forexample, nucleic acids, proteins, peptides, cells, viruses, or bacteria)to interact simultaneously with a test sample with a very high probedensity, it is necessary for all the probes to operate effectively underan identical set of conditions. This is an important limitation forprotein microarrays, in which different protein-protein interactionsonly occur within different solvents, test sample pH, or in the presenceof additional chemicals (such as enzymes).

For this reason, the majority of protein interaction studies arecurrently performed within microtiter plate wells, where each individualwell acts as a separate reaction vessel. By performing proteininteraction studies within microtiter plates, separate chemicalreactions can occur within adjacent wells without intermixing reactionfluids. Therefore, chemically distinct test solutions can be applied toindividual wells. Standard format microtiter plates are available in96-well, 384-well, and 1536-well cartridges. The use of standard formatsfor microtiter plates has enabled manufacturers of high-throughputscreening equipment and instrumentation to build equipment that allaccept the same size cartridge for robotic handling, fluid dispensing,and assay measurement.

One of the embodiments of this invention is a resonant reflectionsurface that can be incorporated into any standard format microtiterplate. The implementation of this embodiment is identical to amicroarray format, except the size of the resonant reflection surface isincreased from a standard microscope slide (˜25×75 mm) to the size of amicrotiter plate (˜3.25×5 inches). Additionally, the resonant reflectionsurface is incorporated into the bottom surface of a microtiter plate byassembling the walls of the reaction vessels over the resonantreflection surface, as shown in FIG. 17, so that each reaction “spot”can be exposed to a distinct test sample. A microtiter plate of theinvention can be used as biosensor and microarray sensor as describedherein.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLE 1

Five circular diffuse grating holograms were prepared by stamping ametal master plate into vinyl. The circular holograms were cut out andglued to glass slides. The slides were coated with 1000 angstroms ofaluminum. In air, the resonant wavelength of the grating is ˜380 nm, andtherefore, no reflected color is visible. When the grating is coveredwith water, a light blue reflection is observed. Reflected wavelengthshifts will only be observable and measurable while the grating iscovered with a liquid, or if a protein film covers the structure.

The purpose of this experiment is to immobilize both proteins andbacteria onto the surface of a grating at high concentration, and tomeasure the wavelength shift induced. For each material, a 20 ul dropletwill be placed onto the active sensor area and allowed to dry in air. At1 ug/ml protein concentration, a 20 ul droplet spreading out to cover a1 cm diameter circle will deposit 2×10⁻⁸ grams of material. The surfacedensity will be 25.6 ng/mm².

-   -   A. HIGH CONCENTRATION PROTEIN IMMOBILIZATION—(slide 4)        -   2% BSA (bovine serum albumin) in DI, 10 ul droplet (0.8 g            BSA in 40 ml DI). Droplet spread out to cover 1 cm diameter            circle.        -   The droplet deposits 0.0002 g of BSA, for a density of            2.5e-6 g/mm{circumflex over ( )}2        -   After protein deposition, Slide 4 appears to have a green            resonance in air.    -   B. BACTERIA IMMOBILIZATION—(slide 2)        -   NECK borrelia Lyme Disease bacteria (1.8e8 cfu/ml, 20 ul            droplet)        -   After bacteria deposition, Slide 2 still looks grey in air    -   C. LOW CONCENTRATION PROTEIN IMMOBILIZATION—(slide 6)        -   0.02% BSA in DI, 10 ul droplet (0.8 g BSA in 40 ml DI).            Droplet spread out to cover a 1 cm diameter circle.        -   The droplet deposits 0.000002 g of BSA for a density of            2.5e-8 g/mm{circumflex over ( )}2.        -   After protein deposition, Slide 6 still looks grey in air

In order to obtain quantitative data on the extent of surfacemodification resulting from the above treatments, the holograms weremeasured at Brown University using SpectraScan system from PhotonicsResearch. The reflectance spectra are presented below.

Because a green resonance signal was immediately visually observed onthe slide upon which high concentration BSA was deposited (Slide 4), itwas measured in air. FIG. 4 shows two peaks at 540 nm and 550 nm ingreen wavelengths where none were present before protein deposition,indicating that the presence of a protein thin film is sufficient toresult in a strong shift in resonant wavelength of a surface reliefstructure.

Because no visible resonant wavelength was observed in air for the slideupon which a low concentration of protein was applied (Slide 6), it wasmeasured with distilled water on surface and compared against a slidewhich had no protein treatment. FIG. 5 shows that the resonantwavelength for the slide with protein applied shifted to the greencompared to a water-coated slide that had not been treated.

Finally, a water droplet containing Lyme Disease bacteria borreliaburgdorferi was applied to a grating structure and allowed to dry in air(Slide 2). Because no visually observed resonance occurred in air afterbacteria deposition, the slide was measured with distilled water on thesurface and compared to a water-coated slide with that had undergone noother treatment. As shown in FIG. 6, the application of bacteria resultsin a resonant frequency shift to longer wavelengths as expected.

EXAMPLE 2

To demonstrate the concept that a resonant grating structure can be usedas a biosensor by measuring the reflected wavelength shift that isinduced when biological material is adsorbed onto its surface, thestructure shown in FIG. 7 was modeled by computer. For purposes ofdemonstration, the substrate chosen was glass (n_(substrate)=1.454)coated with a layer of silicon nitride (t₃=90 μm, n₃=2.02). The gratingis two-dimensional pattern of photoresist squares (t₂=90 nm, n₂=1.625)with a period of 510 nm, and a filling factor of 56.2% (i.e. 56.2% ofthe surface is covered with photoresist squares while the rest is thearea between the squares). The areas between photoresist squares arefilled with a lower refractive index material. The same material alsocovers the squares and provides a uniformly flat upper surface. For thissimulation, a glass layer was selected (n₁=1.45) that covers thephotoresist squares by t₂=100 nm. To observe the effect on the reflectedwavelength of this structure with the deposition of biological material,variable thicknesses of protein (n_(bio)=1.5) were added above the glasscoating layer.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software (Grating Solver Development Company), which utilizesfull 3-dimensional vector code using hybrid. Rigorous Coupled WaveAnalysis and Modal analysis. GSOLVER calculates diffracted fields anddiffraction efficiencies from plane wave illumination of arbitrarilycomplex grating structures. The illumination may be from any incidenceand any polarization.

The results of the computer simulation are shown in FIG. 8 and FIG. 9.As shown in FIG. 10, the resonant structure allows only a singlewavelength, near 805 nm, to be reflected from the surface when noprotein is present on the surface. Because the peak width athalf-maximum is <0.25 nm, resonant wavelength shifts of 1.0 nm will beeasily resolved. FIG. 8 also shows that the resonant wavelength shiftsto longer wavelengths as more protein is deposited on the surface of thestructure. Protein thickness changes of 1 nm are easily observed. FIG. 9plots the dependence of resonant wavelength on the protein coatingthickness. A near linear relationship between protein thickness andresonant wavelength is observed, indicating that this method ofmeasuring protein adsorption can provide quantitative data.

EXAMPLE 3

For a proteomics application, a biosensor array can be operated asfollows: First, a biosensor array surface is prepared with an array ofbait proteins. Next, the biosensor array is exposed to a test samplethat contains a mixture of interacting proteins or a phage displaylibrary, and then this biosensor surface is rinsed to remove all unboundmaterial. The biosensor chip is optically probed to determine whichsites have experienced the greatest degree of binding, and to provide aquantitative measure of bound material. Next, the sensor chip is placedin a “flow cell” that allows a small (<50 microliters) fixed volume offluid to make contact to the sensor chip surface. One electrode isactivated so as to elute bound material from only a selected sensorarray location. The bound material becomes diluted within the flow cellliquid. The flow cell liquid is pumped away from the sensor surface andis stored within a microtiter plate, or some other container. The flowcell liquid is replaced with fresh solution, and a new sensor electrodeis activated to elute its bound material. The process is repeated untilall sensor array regions of interest have been eluted and gathered intoseparate containers. If the sample liquid contained a mixture ofproteins, protein contents within the separate containers can beanalyzed using a technique such as electrospray tandem massspectrometry. If the sample liquid contained a phage display library,the phage clones within the separate containers can be identifiedthrough incubation with a host strain bacteria, concentrationamplification, and analysis of the relevant library DNA sequence.

1-104. (canceled)
 105. An apparatus for detecting the presence andconcentration of matter in contact with a surface structure opticalfilter by observation of a shift in the wavelength of filteredelectromagnetic waves, the apparatus comprising: a first substratehaving a surface relief structure containing at least one dielectricbody with physical dimensions smaller than the wavelength of thefiltered electromagnetic waves, such structures repeated in a lineararray or two dimensional array covering at least a portion of thesurface of the first substrate, said surface relief structures of thesubstrate being composed of or immersed in a material sufficient to forma guided mode resonance filter; and a sample material deposited on thesurface relief structures, thereby producing an observable shift in thewavelength of the filtered electromagnetic waves in proportion to theamount of sample material accumulated.
 106. An apparatus as in claim105, wherein the spacing of the surface relief structures in the arrayis substantially the same and less than the wavelength of the filteredelectromagnetic waves.
 107. An apparatus as in claim 105, wherein theindividual dielectric bodies in the surface texture are circularlyshaped.
 108. An apparatus as in claim 105, wherein the propagationdirection of electromagnetic waves resonantly reflected from the surfacestructures, or transmitted through the substrate, is not materiallyaltered by the accumulation of sample material on the surfacestructures.
 109. An apparatus for detecting the concentration of matterin a material layer by observation of a shift in the wavelength offiltered electromagnetic waves, the apparatus comprising: a substratehaving a surface relief structure containing at least one dielectricbody with physical dimensions smaller than the wavelength of thefiltered electromagnetic waves, such structures repeated in a lineararray or two dimensional array covering at least a portion of thesurface of the substrate; a material coating the surface reliefstructures of the substrate to form a guided mode resonance filter; anda top material layer which adheres or chemically binds to a samplematerial thereby producing an observable shift in the wavelength of thefiltered electromagnetic waves.
 110. An apparatus as in claim 109,wherein the spacing of the surface relief structures in the array issubstantially the same and less than the wavelength of the filteredelectromagnetic waves.
 111. An apparatus as in claim 109, wherein thesurface relief structure is composed of a conductive material suitablefor applying an electric field.
 112. An apparatus as in claim 109,further comprising a second resonant structure coupled to the firstsubstrate to provide a static reference signal which can be used todetermine the difference between a shifted signal due to a depositedmaterial layer and a shifted signal due to varying ambient conditions.113. An apparatus as in claim 109, wherein the individual dielectricbodies comprising the surface texture have cylindrical, elliptical,square, rectangular, or hexagonal cross sectional profiles.
 114. Anapparatus as in claim 109, wherein the individual dielectric bodies inthe surface texture are lines with a width less than the wavelength ofthe filtered electromagnetic waves and a length substantially equivalentto the apparatus dimension, repeated in an array with a spacing lessthan the wavelength of the filtered electromagnetic waves.
 115. Anapparatus as in claim 109, wherein the substrate comprises glass orplastic.
 116. An apparatus as in claim 109, wherein the dielectricbodies comprising the surface relief structures are comprised of amaterial selected from the group consisting of zinc sulfide, titaniumoxide, tantalum oxide and silicon nitride.
 117. An apparatus as in claim109, wherein the sample material comprises an organic substance.
 118. Anapparatus as in claim 109, wherein the sample material comprises aninorganic substance.
 119. An apparatus as in claim 105, wherein theindividual dielectric bodies comprising the surface texture havecylindrical, elliptical, square, rectangular, or hexagonal crosssectional profiles.
 120. An apparatus as in claim 105, wherein in theindividual dielectric bodies comprising the surface texture are lineswith a width less than the wavelength of the filtered electromagneticwaves and length substantially equivalent to the apparatus dimension,repeated in an array with a spacing less than the wavelength of thefiltered electromagnetic waves.
 121. An apparatus as in claim 105,wherein the substrate comprises glass or plastic.
 122. An apparatus asin claim 105, wherein the dielectric bodies comprising the surfacerelief structures are comprised of a material selected from the groupconsisting of zinc sulfide, titanium oxide, tantalum oxide and siliconnitride.
 123. An apparatus as in claim 105, wherein the surface reliefstructure is a conductive material.
 124. An apparatus as in claim 105further comprising a conductive material to allow an electric field tobe applied.
 125. An apparatus as in claim 105, further comprising asecond resonant structure coupled to the first substrate to provide astatic reference signal which can be used to determine the differencebetween a shifted signal due to a deposited material layer and a shiftedsignal due to varying ambient conditions.
 126. An apparatus as in claim105, wherein the sample material comprises an organic substance.
 127. Anapparatus as in claim 105, wherein the sample material comprises aninorganic substance.